The Effect of CCN1 on the Replication of Influenza A virus and Influenza B virus in MDCK Cells
TANG Tian1,2, YANG Chen-Hao1,3, LI Cheng-Fang1,3, HE Ting1,2, XU Ling-Long1,2, LIU Zhen-Bin1,3, QIAO Zi-Lin1,3,*
1 Engineering Research Center of Key Technologies for Cell-Based Vaccines and Industrialization, Ministry of Education/Gansu Provincial Animal Cell Technology Innovation Center, Northwest Minzu University, Lanzhou 730030, China; 2 College of Life Sciences and Engineering, Northwest Minzu University, Lanzhou, 730030, China; 3 Key Laboratory of Bioengineering and Technology, State Ethnic Affairs Commission, Northwest Minzu University, Lanzhou 730030, China
Abstract：Cellular communication network factor 1 (CCN1), an extracellular matrix-associated protein critical for inflammation control and tissue repair, has not yet been functionally characterized in antiviral innate immunity. In this study, Madin-Darby canine kidney (MDCK) cells were infected with Influenza virus to dissect the role of CCN1 during Influenza A virus (IAV) and Influenza B virus (IBV) replication and the underlying signaling pathways. CCN1-knock-down and over-expressing cell lines were established, and viral replication was quantified by qPCR, Western blot and 50% tissue culture infectious dose (TCID50) assays. To trigger innate immune signaling, MDCK cells were stimulated with interferon-β (IFN-β) or low-/high-molecular-weight (LMW/HMW) polyinosinic-polycytidylic acid (Poly(I:C)). Influenza virus infection strongly up-regulated CCN1 transcription: Increases of (8.48±0.17)-fold (H1N1), (9.96±0.31-fold) (BY), (16.37±0.74)-fold (H5N1) and (16.20±0.53)-fold (H3N2) were observed. After treatment with the innate immune signaling pathway activator Poly(I:C)(LMW), the expression of CCN1 was significantly upregulated in MDCK cells. In MDCK cells overexpressing CCN1, the expression levels of Influenza virus nucleoprotein (NP) and non-structural protein 1 (NS1) genes were significantly upregulated following infection. In CCN1-over-expressing cells challenged with IAV, NP and NS1 levels rose significantly, by (4.871±0.02478) and (4.410±0.2532) times, respectively, compared with the control group. MDCK cells with CCN1 knockdown exhibited a significant downregulation in the expression levels of viral NP and NS1 genes following Influenza virus infection. Moreover, CCN1 over-expression suppressed JAK-STAT downstream genes. Collectively, CCN1 regulated innate immunity during Influenza virus infection. This study providing a new strategy for targeted screening of vaccine high-yielding vaccine cell substrates.
唐甜, 杨晨灏, 李成芳, 何婷, 许玲珑, 刘振斌, 乔自林. CCN1对甲型和乙型流感病毒在MDCK细胞中复制的影响[J]. 农业生物技术学报, 2026, 34(6): 1271-1286.
TANG Tian, YANG Chen-Hao, LI Cheng-Fang, HE Ting, XU Ling-Long, LIU Zhen-Bin, QIAO Zi-Lin. The Effect of CCN1 on the Replication of Influenza A virus and Influenza B virus in MDCK Cells. 农业生物技术学报, 2026, 34(6): 1271-1286.
[1] 刘爱军, 张传亮, 安慧婷, 等. 2025. TRIM家族蛋白在甲型流感病毒感染中的作用研究进展[J]. 中国预防兽医学报, 47(1): 108-116. (Liu A J, Zhang C L, An H T, et al.2025. Research progress on the role of TRIM family proteins in Influenza A virus infection[J]. Chinese Journal of Preventive Veterinary Medicine, 47(1): 108-116.) [2] 王思雅. 2025. LncRNA (LOC106559853)抑制流感病毒在MDCK细胞中的复制[D]. 硕士学位论文, 西北民族大学, 导师: 刘振斌, pp. 5. (Wang S Y.2025. LncRNA(LOC106559853) inhibits Influenza virus replication in MDCK cells[D]. Thesis for M. S., Northwest Minzu University, Supervisor: Liu Z B, pp. 5. [3] 张文强, 宋绍霞, 王同展. 2012. 甲型H1N1流感病毒HA蛋白结构和功能研究进展[J]. 病毒学报, 28(04): 444-452. (Zhang W Q, Song S X, Wang T Z.2012. Research progress on structure and function of HA protein of influenza A H1N1 virus[J]. Chinese Journal of Virology, 28(4): 444-452.) [4] Aregger M, Kuglstatter A, D'Arcangelo E, et al.2019. Pooled lentiviral CRISPR-Cas9 screens for functional genomics in mammalian cells[J]. Methods in Molecular Biology, 1869: 169-188. [5] Chang M X.2021. The negative regulation of retinoic acid-inducible geneⅠ (RIG-Ⅰ)-like receptors (RLRS) signaling pathway in fish[J].Developmental and Comparative Immunology, 119: 104038. [6] Chen C C, Lau L F.2009. Functions and mechanisms of action of CCN matricellular proteins[J]. International Journal of Biochemistry & Cell Biology, 41(4): 771-783. [7] Denz P J, Speaks S, Kenney A D, et al.2024. Innate immune control of Influenza virus interspecies adaptation via IFITM3[J]. Nature Communications, 15(1): 9375. [8] Ène M A, Guedon E, Mignee C, et al.2022. From functional genomics of vero cells to CRISPR-based genomic deletion for improved viral production rates[J]. Biotechnology and Bioengineering 119(10): 2794-2805. [9] García-Sastre A, Egorov A, Matassov D, et al.1998. Influenza a virus lacking the NS1 gene replicates in interferon-deficient systems[J]. Virology, 252(2): 324-330. [10] Goraya M U, Wang M, Zheng C, et al.2020. Web of interferon-stimulated antiviral factors to control the Influenza A virus replication[J]. Microbial Pathogenesis, 139: 103919. [11] Hale B G, Randall R E, Ortín J, Jackson D.2008. The multifunctional NS1 protein of Influenza a virus[J]. Journal of General Virology, 89(Pt 10): 2359-2376. [12] Iwasaki A, Pillai P S.2014. Innate immunity to Influenza virus infection[J]. Nature Reviews Immunology, 14(5): 315-328. [13] Kandhaya-Pillai R, Yang X, Tchkonia T, et al.2022. TNF-α/IFN-γ synergy amplifies senescence-associated inflammation and SARS-COV-2 receptor expression via hyper-activated JAK/STAT1[J]. Aging Cell, 21(6): e13646. [14] Kim S M, Park J H, Chung S K, et al.2004. Coxsackievirus B3 infection induces cyr61 activation via JNK to mediate cell death[J]. Journal of Virology, 78(24): 13479-13488. [15] Krischuns T, Lukarska M, Naffakh N, Cusack S.2021. Influenza virus RNA-dependent RNA polymerase and the host transcriptional apparatus[J]. Annual Review of Biochemistry, 90: 321-348. [16] Kurozumi K, Hardcastle J, Thakur R, et al.2008. Oncolytic HSV-1 infection of tumors induces angiogenesis and upregulates CYR61[J]. Molecular Therapy, 16(8): 1382-1391. [17] Lau L F.2011. CCN1/CYR61: The very model of a modern matricellular protein[J]. Cellular and Molecular Life Sciences, 68(19): 3149-3163. [18] Li G, Zhang J, Zhao Z, et al.2024. RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation[J]. EMBO Reports, 25(10): 4594-4624. [19] Liang S, Lin J, Xiao M, et al.2024. Effect of Haoqin Qingdan Tang on Influenza A virus through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway[J]. Phytomedicine, 129: 155680. [20] Liao Y, Wang Y, Li J, et al.2025. USP1 inhibits influenza A and B virus replication in MDCK cells by mediating RIG-Ⅰdeubiquitination[J]. Cellular and Molecular Life Sciences, 82(1): 200. [21] Liu X, Chen H, Ye G, et al.2024. African swine fever virus PB318l, a trans-geranylgeranyl-diphosphate synthase, negatively regulates CGAS-sting and IFNAR-JAK-STAT signaling pathways[J]. PLOS Pathogens, 20(4): e1012136. [22] Mayuramart O, Poomipak W, Rattanaburi S, et al.2022. IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing Influenza virus replication and improving vaccine production[J]. PeerJ, 10: e13989. [23] Mooring M, Yeung G A, Luukkonen P, et al.2023. Hepatocyte CYR61 polarizes profibrotic macrophages to orchestrate nash fibrosis[J]. Science Translational Medicine, 15(715): eade3157. [24] O’Rourke S M, Byrne G, Tatsuno G, et al.2018. Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production[J]. Public Library of Science ONE, 13(8): e0197656. [25] Rieder M, Conzelmann K K.2009. Rhabdovirus evasion of the interferon system[J]. Journal of Interferon and Cytokine Research, 29(9): 499-509. [26] Pan Q, Xie Y, Zhang Y, et al.2024. EGFR core fucosylation, induced by Hepatitis C virus, promotes TRIM40-mediated RIG-Ⅰ ubiquitination and suppresses interferon-Ⅰ antiviral defenses[J]. Nature Communications, 16(1): 8004. [27] Peng Y, Guo J, Sun T, et al.2020. USP39 serves as a deubiquitinase to stabilize STAT1 and sustains typeⅠ IFN-induced antiviral immunity[J]. Journal of Immunology, 205(11): 3167-3178. [28] Philips R L, Wang Y, Cheon H, et al.2022. The JAK-STAT pathway at 30: Much learned, much more to do[J]. Cell, 185(21): 3857-3876. [29] Pizzolla A, Smith J M, Brooks A G, et al.2017. Pattern recognition receptor immunomodulation of innate immunity as a strategy to limit the impact of Influenza virus[J]. Journal of Leukocyte Biology, 101(4): 851-861. [30] Qiao Z, Liao Y, Pei M, et al.2022. RSAD2 is an effective target for high-yield vaccine production in MDCK cells[J]. Viruses, 14(11): 2587. [31] Schenker A J, Ortiz-Hernández G L.2025. CYR61 as a potential biomarker and target in cancer prognosis and therapies[J]. Cells, 14(11): 761. [32] Schoggins J W, MacDuff D A, Imanaka N, et al.2019. Interferon-stimulated genes: What do they all do?[J]. Annual Review of Virology, 6(1): 567-584. [33] Shimoyama T, Asano Y, Kuwahara-Ota R, et al.2010. CCN3 inhibits neointimal hyperplasia through modulation of smooth muscle cell growth and migration[J]. Arteriosclerosis, Thrombosis, and Vascular Biology, 30(4): 675-682. [34] Spalding V A, Li Y, Hadi K, et al.2024. YAP mediates HIV-related liver fibrosis[J]. Journal of Hepatology, 6(11): 101163. [35] Verrecchia F, Mauviel A.2004. TGF-beta and TNF-alpha: Antagonistic cytokines controlling typeⅠ collagen gene expression[J]. Cellular Signalling, 16(8): 873-880. [36] Villarino A V, Kanno Y, O'Shea J J.2017. Mechanisms and consequences of JAK-STAT signaling in the immune system[J]. Nature Immunology,18(4): 374-384. [37] Wang S,Li Z,Chen Y, et al2003. ARIH1 inhibits Influenza A virus replication and facilitates RIG-Ⅰ dependent immune signaling by interacting with SQSTM1/p62[J]. Virology Journal, 20(1): 58. [38] Wegler C, Gazit M, Issa K, et al.2021. Expanding the efflux in vitro assay toolbox: A CRISPR-Cas9-edited MDCK cell line with human BCRP and completely lacking canine MDR1[J]. Journal of Pharmaceutical Sciences 110(1): 388-396. [39] Wiedmaier N, Müller S, Köberle M, et al.2008. Bacteria induce CTGF and CYR61 expression in epithelial cells in a lysophosphatidic acid receptor-dependent manner[J]. Journal of Medical Microbiology, 298(3-4): 231-243. [40] Wu X, Wang J, Wang S, et al.2019. Inhibition of Influenza A virus replication by TRIM14 via its multifaceted protein-protein interaction with NP[J]. Frontiers in Microbiology, 10: 344. [41] Yan Z, Gibson S A, Buckley J A, et al.2018. Role of the JAK/STAT signaling pathway in regulation of innate immunity in neuroinflammatory diseases[J]. Clinical Immunology, 189: 4-13. [42] Yang Z, Yu S, Xu Y, et al.2024. The screening and mechanism of influenza-virus-sensitive MDCK cell lines for influenza vaccine production[J]. Diseases, 12(1): 18. [43] Zhang J, Zhao R Y, Ejikemeuwa A, et al.2022. Understanding the role of SARS-CoV-2 ORF3a in viral pathogenesis and COVID-19[J]. Frontiers in Microbiology, 13: 854567.
